FPLC

(Fast protein liquid chromatography) or

(Fast performance liquid
chromatography)
Contents …
• Introduction
• Principle
• Instrumentation
• Advantages
• Drawbacks
• Application
• Conclusion
Introduction
• FPLC is basically a “protein friendly” HPLC
system.
• FPLC is an intermediate between classical
column chromatography and HPLC.
• It is a complete system for
chromatographic separations of proteins and
other biomolecules such as nucleic acids.
• It is commonly used in biochemistry and
enzymology .
Principle

1. Size(size exclusion chromatography)

2. Charge distribution(ion exchange)
3. Affinity chromatography
1. Size (size exclusion chromatography)
separate proteins according to their size. Also
termed as “Gel Filtration Chromatography”
2. Charge distribution(ion exchange)
separate proteins based on surface-
charges.
3. Affinity chromatography
separate protein based on a highly specific
interaction such as that between antigen
and antibody, enzyme and substrate, or
receptor and ligand.
Instrumentation
Stationary Phase : It is typically a resin composed of
cross-linked agarose beads with varying surface ligands.
Mobile Phase : Mostly buffers, organic solvents.

Pump : Constant controlled flow by peristaltic pumps.

The flow rate is varied based on scale of preparation ie;
analytical or preparative chromatography.
Mixer: Powered and controlled by the pump. Especially
important when forming gradients between buffer
sources. The mixer ensures that the buffers used are in
the correct proportion relative to each other during the
course of the FPLC run.
Instrumentation
Injection valve:
• Pumps are connected to valves which send the buffers in the
desired direction.
• The Inject position is for injecting contents of the sample
loop onto a column.
➢ Column:
• large [internal diameter, mm] tubes.
• contains small [ 13–15 µ] particles or gel beads.
• Typical column materials are inert plastic or inert fluid surfaces
like Teflon, titanium, and glass.
• It is designed to operate at not more than 580 psi. Pre-packed
• columns are also available.
• Columns should be stored in 24% ethanol/H2O when not in
use.
Instrumentation
Fraction collector: Allows fixed volume fractionation.

Flow Restrictor: Generates a steady back-pressure to

prevent air bubbles being formed after the columns in
the flow cells.
On-line Filter: Rejects sample particulates that may
clog the fluidic system by generating a maximum back-
pressure of 0.5 MPa.
Detection system: UV or UV/Vis spectrophotometer,
Conductivity detector and Refractive Index (RI) Detector
based on characteristics of the analyte of interest.
Instrumentation
Instrumentation
How does it differ from HPLC ??

FPLC differs from HPLC in that the columns used

for FPLC can only be used up to maximum pressure of 3-
4 MPa (435-580 psi).
Stainless steel components replaced with glass and
plastic. Inert surfaces are necessary since many of
the resolving buffers contain high concentrations of
halide salts that attack and corrode stainless steel
surfaces.
FPLC pump delivers a solvent flow rate in the range of 1-
499ml/hr HPLC pump gives a rate 0.010-10ml/min.
FPLC system can use FPLC columns only but in
Advantages
• Reproducible with excellent resolution.
• Very simple system programming.
• Inert construction against the very high salt
concentrations and corrosive liquids hence
columns have longer lifetime.
•Since lower pressures are used in FPLC than in
HPLC, a wider range of column supports are
possible.
•The wide flow range makes it suitable both
for analytical and preparative
chromatography.
Drawbacks
• Needs glass columns.
• Can not handle high pressure.
• Instrument does not support HPLC columns.
• Purifying thermo labile (heat sensitive)
proteins is a tough task.
Application
• Protein analysis.
• Lipoprotein separation by FPLC.
• Purification of animal venoms.
• Separation of Plasma Proteins in Urine and Cerebrospinal
Fluid.
• Isolation of porphobilinogen deaminase from human
erythrocytes
• Rapid purification of RNA.
• The Analysis of nitrogenous constituents of beer.
• Coupled to a double focusing inductively coupled plasma mass
spectrometer for trace metal speciation in human serum and
detectable levels of Cr, Al, Pb and Sn were present in uraemic
sera.
• FPLC method could be used as a cost-effective method for
routine β-thalassaemia diagnosis.
Thank You
….